Incucyte s3 real time cell imaging system

To study cell invasion in culture, cSCC cells were transfected with negative control and BRD3OS (LINC00094), MMP-1, or MMP-13 targeting siRNAs (75 nM) and cultured for 24 h. Transfected cells were plated on a collagen type I–coated (5 μg/cm², PureCol; Advanced BioMatrix, San Diego, CA, USA) ImageLock 96-well plate (Essen Bioscience, Ann Arbor, MI, USA), and cells were allowed to adhere overnight. The cell monolayer was scratched using an Incucyte wound maker (Essen Bioscience), and collagen type I solution was added by mixing type I collagen (PureCol) with 5× Dulbecco’s Modified Eagle Medium and 0.2 mol/L HEPES buffer (pH 7.4) at a ratio of 7:2:1, respectively. Finally, 1 mol/L NaOH was added to obtain a final pH of 7.4. Collagen was allowed to polymerize for 2 h at 37 °C, and a cell culture medium with 0.5% fetal calf serum was added on top. The gap closure was imaged using the IncuCyte S3 real-time cell imaging system (Essen Bioscience), and the relative cell invasion was quantitated using the IncuCyte S3 software version 2020A (Essen Bioscience).

Specific small-molecule factor D inhibitor danicopan (ACH-4471, MedChemExpress (Monmouth Junction, NJ, USA)) was used to inhibit the activity of FD in cSCC cultures [18 (link),41 (link)]. Cultured cSCC cells (UT-SCC-12A, UT-SCC-91, UT-SCC-59A, UT-SCC-105) were plated on 96-well plates (4000 cells/well) in the presence of 10% fetal calf serum. After overnight incubation, the small-molecule FD inhibitor danicopan (ACH-4471) was added to the wells in serum-free medium in different concentrations (0.1 µM, 1 µM and 10 µM). DMSO was added to control cultures as a vehicle control. Cell proliferation was determined with the IncuCyte S3 real-time cell imaging system (Essen Bioscience, Ann Arbor, MI, USA). Analysis of the results was performed using IncuCyte S3 software (Essen Bioscience, Ann Arbor, MI, USA). Relative confluence was measured as an index of cSCC cell proliferation.